Sang-Keun Kim

Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence.

Reactive oxygen species (ROS) were generated in all oxygen‐utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II−/− mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II−/− MEF. Senescence‐associated (SA)‐β‐galactosidase (Gal)‐positive cell formation was about 30% higher in Prx II−/− MEF. N‐Acetyl‐l‐cysteine (NAC) treatment attenuated SA‐β‐Gal‐positive cell formation. Prx II−/− MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.4% in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII−/− MEF cells. The cellular senescence of Prx II−/− MEF was correlated with the organismal aging of Prx II−/− mouse skin. While extracellular signal‐regulated kinase (ERK) and p38 activation was detected in Prx II−/− MEF, ERK and c‐Jun N‐terminal kinase (JNK) activation was detected in Prx II−/− skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.

Korean Red Ginseng Saponin Fraction Downregulates Proinflammatory Mediators in LPS Stimulated RAW264.7 Cells and Protects Mice against Endotoxic Shock

Korean red ginseng has shown therapeutic effects for a number of disease conditions. However, little is known about the antiinflammatory effect of Korean red ginseng saponin fraction (RGSF) in vitro and in vivo. Therefore, in this study, we showed that RGSF containing 20(S)-protopanaxadiol type saponins inhibited nitric oxide production and attenuated the release of tumor necrotic factor (TNF)-α, interleukin (IL)-6, granulocyte monocyte colony stimulating factor (GMCSF), and macrophage chemo-attractant protein-1 in lipopolysaccharide (LPS) stimulated murine macrophage RAW264.7 cells. Moreover, RGSF down-regulated the mRNA expressions of inducible nitric oxide synthase, cyclooxyginase-2, IL-1β, TNF-α, GMCSF, and IL-6. Furthermore, RGSF reduced the level of TNF-α in the serum and protected mice against LPS mediated endotoxic shock. In conclusion, these results indicated that ginsenosides from RGSF and their metabolites could be potential sources of therapeutic agents against inflammation.

Phellinus baumii ethyl acetate extract inhibits lipopolysaccharide-induced iNOS, COX-2, and proinflammatory cytokine expression in RAW264.7 cells.

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT–PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E2 (PGE2) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1β, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

Hepatitis B Virus X Protein Regulates Hepatic Glucose Homeostasis via Activation of Inducible Nitric Oxide Synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Vitrification of Canine Oocytes

ABSTRACT The objective of the present study was to compare the vitrification method for cryopreservation of canine oocytes. Canine cumulus-oocyte complexes (COCs) were collected from ovaries, and were vitrified by ethylene glycol based (E30S) or DMSO based (DAP213) methods. In the E30S method, COCs were exposed to the vitrification solution, composed of 30% ethylene glycol and 0.5 M sucrose, step-wise transferred onto a cryotop holder, then plunged directly into liquid nitrogen. In the DAP213 method, COCs were exposed to 1 M DMSO and DAP213 solution in a cryotube, and thereafter plunged directly into liquid nitrogen. Although vitrified-warmed COCs in the E30S method showed fewer morphological abnormalities, and higher viability than those in the DAP213 method, there was no significant difference in between. These results indicate that either method of vitrification is available and statistically comparable for cryopreservation of canine oocytes.

Transgenic expression of Korean type hepatitis C virus core protein and related mutants in mice

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S116I, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S116I transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.

Mechanism of anti-platelet activity of Oligoporus tephroleucus oligoporin A: Involvement of extracellular signal-regulated kinase phosphorylation and cyclic nucleotide elevation

This study investigated the inhibitory effects of oligoporin A on platelet aggregation and the mechanism of its action on downstream signaling molecules. Oligoporin A was isolated from the fruiting bodies of Oligoporus tephroleucus (Polyporaceae). The anti-platelet activities of oligoporin A were studied using rat platelets. The effects of oligoporin A on intracellular Ca2+ mobilization, ATP release, production of the cyclic nucleotides cAMP and cGMP, extracellular signal-regulated kinase (ERK) 2 phosphorylation, and fibrinogen binding to active integrin αIIbβ3 were assessed. Oligoporin A, but not oligoporins B and C, inhibited collagen-induced platelet aggregation in a concentration-dependent manner. Interestingly, oligoporin A did not affect ADP- and thrombin-induced platelet aggregations, which act on different types of membrane receptors. Granule secretion analysis demonstrated that oligoporin A significantly and dose-dependently reduced collagen-induced ATP release and intracellular Ca2+ mobilization. Additionally, oligoporin A induced the dynamic increase in cAMP and cGMP. Increased cGMP production was further confirmed by the simultaneous production of nitric oxide. Pretreatment with oligoporin A significantly blocked collagen-induced ERK2 phosphorylation. Finally, oligoporin A vaguely diminished the binding of fibrinogen to its cognate receptor, integrin αIIbβ3. The results indicate that oligoporin A inhibits only collagen-induced platelet aggregation mediated through the modulation of downstream signaling molecules. Oligoporin A may be beneficial against cardiovascular disease provoked by aberrant platelet activation.

Interaction of Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) and Vav3 in The Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.